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. 2007 Nov 21;129(46):14224-31.
doi: 10.1021/ja074557r. Epub 2007 Oct 30.

CD and MCD of CytC3 and taurine dioxygenase: role of the facial triad in alpha-KG-dependent oxygenases

Michael L Neidig  1 , Christina D Brown, Kenneth M Light, Danica Galonić Fujimori, Elizabeth M Nolan, John C Price, Eric W Barr, J Martin Bollinger Jr, Carsten Krebs, Christopher T Walsh, Edward I Solomon
Affiliations

CD and MCD of CytC3 and taurine dioxygenase: role of the facial triad in alpha-KG-dependent oxygenases

Michael L Neidig et al. J Am Chem Soc. .

Abstract

The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

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Figures

Figure 1
Figure 1
The 298K CD spectra of (A) apoCytC3 (black), apoCytC3/Cl- + 2 equiv FeII (green) and CytC3/FeII/Cl-/α-KG (blue) and (C) CytC3/FeII/Cl-/α-KG/L-Aba-S-CytC2. The low-temperature, 7T MCD spectra of (B) CytC3/FeII/Cl-/α-KG and (D) CytC3/FeII/Cl-/α-KG /L-Aba-S-CytC2. VTVH data (symbols) and their best fit (lines) for (E) CytC3/FeII/Cl-/α-KG collected at 7505 cm-1 and (F) CytC3/FeII/Cl-/α-KG /L-Aba-S-CytC2 collected at 7225 cm-1.
Figure 2
Figure 2
The low temperature, 7T UV/Vis MCD spectra of (A) CytC3/FeII/Cl-/α-KG (blue) and CytC3/FeII/Cl-/α-KG /L-Aba-S-CytC2 (red) and (B) TauD/FeII/α-KG (blue) and TauD/FeII/α-KG/taurine (red).
Figure 3
Figure 3
The 298K CD spectra of (A) apoTauD (black), TauD/FeII (green) and TauD/FeII/α-KG (blue) and (C) TauD/FeII/α-KG/taurine. The low-temperature, 7T MCD spectra of (B) TauD/FeII (green) and TauD/FeII/α-KG (blue) and (D) TauD/FeII/α-KG/taurine.
Figure 4
Figure 4
The low-temperature, 7T MCD spectra of CS2/FeII/α-KG (red), TauD/FeII/α-KG (green) and CytC3/FeII/Cl-/α-KG.
Figure 5
Figure 5
Geometry optimized structures of TauD/FeII/α-KG without and with H-bonding to carboxylate.
See this image and copyright information in PMC

References

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